Project summary Sepsis is a clinical syndrome with life-threatening organ dysfunction that is caused by a dysregulated host response to infection. Cardiovascular collapse induced by cardiac dysfunction and profound vasodilatation represents a main feature of septic shock and contributes to its high mortality. Innate immune activation represents the first line of host defense, but dysregulated innate immune response to pathogens and host danger molecules proves to be deleterious in sepsis. To identify the host danger molecules, we have recently found a significant rise in plasma cell-free RNA in sepsis. These extracellular (ex) RNAs are released from the host cells as well as from invading bacteria and closely correlated with sepsis severity. Plasma miRNA array identifies six miRNAs that are elevated in response to sepsis; four of them (miR-34a, -122, -145, 146a) induce marked cytokine production and complement activation. Moreover, employing genetic deletion models and pharmacological inhibitor, we demonstrate that miRNA mimics activates inniate immune response via specific TLR7 signaling. Finally, systemic TLR7 deficiency attenuates plasma IL-6 production and confers a survival benefit in sepsis. The over goal of this proposal is to determine the role of ex- miRNA-TLR7 signaling in host innate immune activation and in cardiac bioenergetic dysfunction in sepsis. The proposal is based on the following three hypotheses: 1) that host miRNAs are released from injured cells and play an important role in systemic innate immune activation and cardiac bioenergetic dysfunction in sepsis, 2) that ex-miRNAs act through endosomal TLR7 signaling, 3) that pharmacological blockade of miRNA-TLR7 signaling after polymicrobial infection will attenuate the systemic innate immune activation and offer cardiac and survival benefit in sepsis. In Specific Aim 1, we will determine the role of ex-miRNA-TLR7 signaling in mediating systemic innate immune responses in sepsis. In Specific Aim 2, we will define the impact of ex-miRNA-TLR7 signaling to sepsis-induced cardiac energetic dysfunction. In Specific Aim 3, through a series of proof-of-concept pre-clinical studies, we will determine the therapeutic efficacy of synthetic anti-miR oligonucleotide inhibitors and TLR7 antagonist to modulate organ inflammation, organ (liver and kidney) injury, and mortality in sepsis. This proposal addresses a unique function of endogenous ex-miRNA?TLR7 signaling in host innate immune activation and organ injury/dysfunction with a significant implication to sepsis pathogenesis and treatment, and miRNA biology.